Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay
Blood-brain barrier (BBB) P-glycoprotein activity is rapidly reduced by vascular endothelial growth factor (VEGF) acting via Src and by tumor necrosis factor-alpha acting via protein kinase C (PKC)beta1. To probe underlying mechanism(s), we developed an in vivo, immunoblot-based proteinase K (PK) protection assay to assess the changes in the P-glycoprotein content of the BBB's luminal membrane. Infusion of PK into the brain vasculature selectively cleaved luminal membrane P-glycoprotein, leaving intracellular proteins intact. Intracerebroventricular injection of VEGF partially protected P-glycoprotein from proteolytic cleavage, consistent with transporter internalization. Activation of PKCbeta1 did not protect P-glycoprotein. Thus, VEGF and PKCbeta1 reduce P-glycoprotein activity by distinct mechanisms.
Hawkins, B., Rigor, R., & Miller, D. S. (2010). Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay. Journal of Cerebral Blood Flow and Metabolism, 30(9), 1593 - 1597. https://doi.org/10.1038/jcbfm.2010.117